TGFβ1 (Transforming growth factor beta 1) is a multifunctional cytokine regulating growth, migration, differentiation, and apoptosis of diverse epithelial and hematopoietic cells. The pleiotropic effects of TGFβ1 allow it to function as a ‘Janus-faced’ cytokine during carcinogenesis, promoting both tumor growth suppression and malignant progression. Loss of TGFβ signaling as a whole is observed in many cancers, reflecting a role in tumor suppression. On the other hand, cancer cells frequently exhibit selective loss of anti-proliferative response to TGFβ1 without hindering other functions of TGFβ signaling that are advantageous for tumor development such as angiogenesis, invasion, and metastasis.
One of the mechanisms used by cancers to escape the anti-proliferative response to TGFβ1 involves high-level accumulation of c-Myc in cancer cells. Rapid down-regulation of c-Myc is a key event in the TGFβ1-mediated anti-proliferative response in normal epithelial cells, as it is a prerequisite for subsequent induction of key tumor suppressor genes such as p15Ink4b and/or p21cip1. Conversely, aberrant up-regulation of c-Myc is frequently observed in several cancers, irrespective of the TGFβ1 level. Loss of c-Myc repression is sufficient to overcome the anti-proliferative effects of TGFβ1 in breast and ovarian cancers (Chen C R et al., Proc Natl Acad Sci USA 98:992-999, 2001). Therefore, de-regulation of c-Myc expression appears critical for the selective loss of the anti-proliferative response to TGFβ1 in cancers.
Previous studies have shown that down-regulation of c-Myc by TGFβ1 is achieved at the transcriptional level via binding of Smad3 to a silencer element in the c-Myc promoter (Frederick J P et al., Mol Cell Biol 24:2546-2559, 2004), and also at the post-transcriptional level, since c-Myc mRNA is unusually unstable and rapidly degraded in response to TGFβ1 (Coffey R J et al., Mol Cell Biol 8:3088-3093, 1988). However, the detailed mechanisms for the aberrant up-regulation of c-Myc in cancers in the presence of TGFβ1 signaling remain to be established. Recently, Marderosian et al. reported that c-Myc mRNA stability is negatively regulated by binding of TTP (Tristetraprolin) to an AU-rich element (ARE) within 3′-UTR (Marderosian M et al., Oncogene 25:6277-6290, 2006). TTP is induced by various stimuli, including TGFβ1, and is thus a good candidate as a critical posttranscriptional regulator of c-Myc in response to TGFβ1 in cancers.
TTP (Tristetraprolin, ZFP36, TIS11 or NUP475) is a posttranscriptional regulator that functions in the control of inflammatory responses. TTP binds to the ARE of target mRNAs via CCCH tandem zinc finger motifs and destabilizes cytokine and protooncogene transcripts by promoting deadenylation, decapping and exonucleolytic decay. Moreover, TTP functions as a putative tumor suppressor, since its overexpression in various human cell lines promotes apoptosis and delays tumor formation in IL-3-producing tumor model mice. It is possible that the tumor suppressive function of TTP associated with the mRNA destabilizing activity is disrupted in cancers. However, its role in cancer development and progression has not been clearly defined to date.
The present inventors found that TTP is frequently down-regulated in hepatocellular carcinoma (HCC) via specific methylation at a single CpG site located within the TGFβ-responsive region of its promoter. The single CpG site methylation was sufficient to block TGFβ1-mediated TTP induction and subsequent c-Myc down-regulation, thereby suppressing the anti-proliferative effect of TGFβ1 on tumor cells. Based on these results, the present inventors propose that loss of c-Myc posttranscriptional regulation via single CpG site methylation in the TTP promoter shifts TGFβ1 signaling from cell cycle arrest to proliferation in HCC.